Project Summary/Abstract In the initial 5 years of INIA-Neuroimmune funding, we developed a novel assay for binge-like drinking, where mice are offered limited access to 20% ethanol (EtOH) in water early during their circadian dark period. The Drinking in the Dark (DID) assay has been widely adopted and used to probe mechanisms of binge-like drinking. During the ensuing 10 years, we selectively bred two lines of mice for High Drinking in the Dark (HDID-1 and HDID-2), by mating individuals reaching the highest blood alcohol levels (BALs) after a second daily DID session. HDID mice achieve BALs between 170 and 150 mg% after 32 (HDID-1) and 25 (HDID-2) selected generations, respectively. Binge-like drinking is a strong predictor of alcohol use disorder diagnosis and has deleterious health consequences. These models represent genetic risk for high binge drinking. Binge- like drinking has to date been used only infrequently to screen candidate drugs. Recent studies suggest that HDID mice respond to treatment with some, but not all drugs known to reduce drinking (e.g., acamprosate but not naltrexone). Because HDID mice appear to represent a selective model for screening, we propose to use them to nominate and then test INIA-N targets. In Specific Aim 1, we will collaborate with INIA-N PIs Drs. Mayfield, Ponomarev, and Farris to test novel neuroimmune-related targets suggested by their bioinformatics analyses and rigorously test their efficacy in HDID mice. The Mayfield group will compare expression profiles between HDID mice and their non-selected HS/Npt control line with data from the Library of Integrated Network-based Cellular Signatures (LINCS), which comprises gene expression response profiles to >1000 perturbagens in multiple cell lines, including responses to many FDA-approved compounds. We hypothesize that comparing the HDID vs HS gene expression differences with perturbagen-generated difference in LINCS and seeking novel drugs that normalize HDID expression differences from HS will successfully predict drug efficacy to reduce binge-like drinking. Preliminary testing has been carried out successfully for two targets (phosphodiesterase type 4 inhibitor and Bruton's tyrosine kinase inhibitor). For drugs passing these screens, our Specific Aim 2 will extend our efficacy tests of successful drugs to reduction of stress-induced escalations of post-dependence drinking. Through this collaborative Specific Aim with INIA-Stress PIs Drs. Becker and Lopez, we will test the reproducibility and generalization of drug effects in another drinking model, genotype and laboratory. In Specific Aim 3, we will identify the brain localization of novel drug effects and determine their cell type specificity using viral mediated RNAi in collaboration with INIA-N PI Dr. Lasek. We predict that some neuroimmune targets will show neuronal specificity while others will selectively target glial cells. Aim 3 will also measure the drug's physiological effects through collaborations with INIA-PIs Drs. Roberto and Morrisett, who will test specific hypotheses about neuronal excitability. We will continue to provide data to Dr. Mayfield's group, and animals to INIA-N PIs Drs. Hitzemann/Iancu, and other laboratories for functional studies.